Replication-defective retroviral vectors as gene transfer vehicles provide the foundation for human gene therapy. Retroviral vectors are engineered by removing or altering all viral genes so that no viral proteins are made in cells infected with the vector and no further virus spread occurs. The development of packaging cell lines which are required for the propagation of retroviral vectors were the most important step toward the reality of human gene therapy. The foremost advantages of retroviral vectors for gene therapy are the high efficiency of gene transfer and the precise integration of the transferred genes into cellular genomic DNA. However, major disadvantages are also associated with retroviral vectors, namely, the inability of retroviral vectors to transduce non-dividing cells and the potential insertional mutagenesis.
Human adenoviruses have been developed as live viral vaccines and provide another alternative for in vivo gene delivery vehicles for human gene therapy Graham & Prevec in New Approaches to Immunological Problems, Ellis (ed), Butterworth-Heinemann, Boston, Mass., pp. 363-390 (1992) Rosenfeld, et al, Science 252: 431-434 (1991), Rosenfeld, et al, Cell 68: 143-155 (1992), and Ragot, et al, Nature 361: 647-650 (1993)!. The features which make recombinant adenoviruses potentially powerful gene delivery vectors have been extensively reviewed Berkner, Biotechniques 6: 616-629, (1988) and Kozarsky & Wilson, Curr. Opin. Genet. Dev. 3: 499-503, (1993)!. Briefly, recombinant adenoviruses can be grown and purified in large quantities and efficiently infect a wide spectrum of dividing and non-dividing mammalian cells in vivo.
Moreover, the adenoviral genome may be manipulated with relative ease and accommodate very large insertions of DNA.
The first generation of recombinant adenoviral vectors currently available have a deletion in the viral early gene region 1 (herein called E1 which comprises the E1a and E1b regions from genetic map units 1.30 to 9.24) which for most uses is replaced by a transgene. A transgene is a heterologous or foreign (exogenous) gene that is carried by a viral vector and transduced into a host cell. Deletion of the viral E1 region renders the recombinant adenovirus defective for replication and incapable of producing infectious viral particles in the subsequently infected target cells Berkner, Biotechniques 6: 616-629 (1988)!. The ability to generate E1-deleted adenoviruses is based on the availability of the human embryonic kidney packaging cell line called 293. This cell line contains the E1 region of the adenovirus which provides the E1 region gene products lacking in the E1-deleted virus Graham, et al, J. Gen Virol. 36: 59-72, (1977)!. However, the inherent flaws of current first generation recombinant adenoviruses have drawn increasing concerns about its eventual usage in patients. Several recent studies have shown that E1 deleted adenoviruses are not completely replication incompetent Rich, Hum. Gene. Ther. 4: 461-476 (1993) and Engelhardt, et al, Nature Genet. 4: 27-34 (1993)!. Three general limitations are associated with the adenoviral vector technology. First, infection both in vivo and in vitro with the adenoviral vector at high multiplicity of infection (abbreviated m.o.i.) has resulted in cytotoxicity to the target cells, due to the accumulation of penton protein, which is itself toxic to mammalian cells (Kay, Cell Biochem. 17E: 207 (1993)3. Second, host immune responses against adenoviral late gene products, including penton protein, cause the inflammatory response and destruction of the infected tissue which received the vectors Yang, et al, Proc. Natl, Acad. Sci. USA 91: 4407-4411 (1994)!. Lastly, host immune responses and cytotoxic effects together prevent the long term expression of transgenes and cause decreased levels of gene expression following subsequent administration of adenoviral vectors Mittal, et al, Virus Res.28: 67-90 (1993)!.
In view of these obstacles, further alterations in the adenoviral vector design are required to cripple the ability of the virus to express late viral gene proteins, decreasing host cytotoxic responses and the expectation of decreasing host immune response. Engelhardt et al recently constructed a temperature sensitive (ts) mutation within the E2A-encoded DNA-binding protein (DBP) region of the E1-deleted recombinant adenoviral vector Engelhardt, et al, Proc. Natl. Acad. Sci. USA 91: 6196-6200 (1994)! which fails to express late gene products at non-permissive temperatures in vitro. Diminished inflammatory responses and prolonged transgene expression were reported in animal livers infected by this vector (Engelhart, et al 1994). However, the ts DBP mutation may not give rise to a full inactive gene product in vivo, and therefore be incapable of completely blocking late gene expression. Further technical advances are needed that would introduce a second lethal deletion into the adenoviral E1-deleted vectors to completely block late gene expression in vivo. Novel packaging cell lines that can accommodate the production of second (and third) generation recombinant adenoviruses rendered replication-defective by the deletion of the E1 and E4 gene regions hold the greatest promise towards the development of safe and efficient vectors for human gene therapy. The present invention provides for such packaging cell lines and resultant mutant viruses and recombinant viral vectors (for example, adenoviral or AAV-derived) carrying the transgene of interest.